2012 mechanisms of coronavirus cell entry mediated by the viral spike protein
TRANSCRIPT
Viruses 2012, 4, 1011-1033; doi:10.3390/v4061011
virusesISSN 1999-4915
www.mdpi.com/journal/viruses
Review
Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein
Sandrine Belouzard 1, Jean K. Millet 2, Beth N. Licitra 2 and Gary R. Whittaker 2,*
1 Center for Infection and Immunity of Lille, CNRS UMR8204, INSERM U1019, Institut Pasteur de
Lille, Université Lille Nord de France, 59000 Lille, France; E-Mail: [email protected] 2 Department of Microbiology and Immunology, Cornell University, Ithaca, NY 14853, USA;
E-Mails: [email protected] (J.K.M.); [email protected] (B.N.L.)
* Author to whom correspondence should be addressed; E-Mail: [email protected];
Tel.: +1-607-253-4021; Fax: +1-607-253-3384.
Received: 8 May 2012; in revised form: 13 June 2012 / Accepted: 14 June 2012 /
Published: 20 June 2012
Abstract: Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the
cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their
envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and
is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion
protein, and is responsible for binding to the receptor on the host cell as well as mediating
the fusion of host and viral membranes—A process driven by major conformational
changes of the S protein. This review discusses coronavirus entry mechanisms focusing on
the different triggers used by coronaviruses to initiate the conformational change of the S
protein: receptor binding, low pH exposure and proteolytic activation. We also highlight
commonalities between coronavirus S proteins and other class I viral fusion proteins, as
well as distinctive features that confer distinct tropism, pathogenicity and host interspecies
transmission characteristics to coronaviruses.
Keywords: coronavirus; spike; viral entry; fusion; proteolytic activation
OPEN ACCESS
Viruses 2012, 4
1012
1. Introduction
Although the first member of the coronavirus family was discovered in the 1930s [1] coronaviruses
gained particular notoriety when the severe acute respiratory syndrome (SARS) outbreak shook the
world in 2002–2003. Interest in this family of viruses grew in the aftermath of this epidemic, leading to
the identification of many new family members. This episode also shed light on the capabilities of
coronaviruses to jump across species. Before gaining importance for public health in 2003, the
diseases associated with coronaviruses were mainly of veterinary interest. Coronaviruses infect a wide
variety of mammals and birds, causing respiratory and enteric diseases and, in some rarer cases,
hepatitis and neurologic disease. Infection can be acute or persistent [2].
Coronaviruses are classified in four different genera, historically based on serological analysis and
now on genetic studies: alpha-, beta-, gamma-, and delta-CoV (Table 1). Coronaviruses belong to the
Coronavirinae subfamily that together with Torovirinae form the Coronaviridae family in the
Nidovirales order.
Coronaviruses are enveloped, spherical or pleiomorphic viruses, with typical sizes ranging from 80
to 120 nm. They possess a 5' capped, single-strand positive sense RNA genome, with a length between
26.2 and 31.7 kb, the longest amongst all RNA viruses. The genome is composed of six to ten open
reading frames (ORFs). The first ORF comprises two-thirds of the genome and encodes the replicase
proteins, whereas the last third contains the structural protein genes in a fixed order: (HE)-S-E-M-N
(Figure 1A). Variable numbers of ORF encoding accessory proteins are present between these genes.
The genome is packaged into a helical nucleocapsid surrounded by a host-derived lipid bilayer. The
virion envelope contains at least three viral proteins, the spike protein (S), the membrane protein (M)
and the envelope protein (E) (Figure 1B). In addition, some coronaviruses also contain a
hemagglutinin esterase (HE). Whereas the M and E proteins are involved in virus assembly, the spike
protein is the leading mediator of viral entry. The spike protein is also the principal player in
determining host range [3,4].
Viral entry relies on a fine interplay between the virion and the host cell. Infection is initiated by
interaction of the viral particle with specific proteins on the cell surface. After initial binding of the
receptor, enveloped viruses need to fuse their envelope with the host cell membrane to deliver their
nucleocapsid to the target cell. The spike protein plays a dual role in entry by mediating receptor
binding and membrane fusion. The fusion process involves large conformational changes of the spike
protein. Coronaviruses use a variety of receptors and triggers to activate fusion, however fundamental
aspects that enable this initial step of the viral life cycle are conserved. In this review, we will address
entry strategies of coronaviruses and how these mechanisms are related to host tropism and pathogenicity.
Viruses 2012, 4
1013
Table 1. Coronavirus genera, species and host receptor usage.
Genus Species Receptor
Alphacoronavirus
Alphacoronavirus 1 comprising:
Feline Coronavirus (FCoV) serotype 2 Aminopeptidase N
Canine Coronavirus (CCoV) serotype 2 Aminopeptidase N
Transmissible gastroenteritis virus (TGEV) Aminopeptidase N
Human coronavirus 229E Aminopeptidase N
Human coronavirus NL63 ACE2
Porcine Epidemic Diarrhea Coronavirus (PEDV) Aminopeptidase N
Rhinolophus bat coronavirus HKU2
Scotophilus bat coronavirus 512/05
Miniopterus bat coronavirus 1
Miniopterus bat coronavirus HKU8
Betacoronavirus
Betacoronavirus 1 comprising:
Bovine coronavirus (BCoV) Neu 5,9 Ac2
Human coronavirus OC43 (HCoV-OC43) Neu 5,9 Ac2
Equine coronavirus (ECoV)
Human enteric coronavirus (HECoV)
Porcine haemagglutinating encephalomyelitis virus (PHEV)
Canine respiratory coronavirus (CrCoV)
Murine coronavirus comprising:
Existing species of mouse hepatitis virus (MHV)
Rat coronavirus
Puffinosis virus
CEACAM1
Human coronavirus HKU9
Rousettus bat coronavirus HKU4
Tylonycteris bat coronvirus HKU5
SARSr-CoV (SARS related Coronavirus) comprising
Human SARS-CoV
Rhinolophus bat viruses
ACE2
Gamma-
coronavirus
Avian coronavirus comprising:
IBV
Various coronaviruses infecting turkey, pheasant, duck, goose
and pigeon
Beluga Whale coronavirus SW1
Delta-coronavirus
Bulbul coronavirus HKU11
Thrush coronavirus HKU12
Munia coronavirus HKU13
Viruses 2012, 4
1014
Figure 1. Coronavirus genomes (A). The genome of four different coronaviruses is
depicted. The open reading frame (ORF)1a/b is colored in red. The HE gene present in
MHV-A59 is represented in purple. The gene of structural proteins S (blue), E (pink), M
(dark pink) and N (cyan) are localized in the 3' part of the genome. ORFs encoding
accessory proteins are represented in grey. Coronavirus virion structure (B).
2. Spike Protein
The spike protein is a large type I transmembrane protein ranging from 1,160 amino acids for avian
infectious bronchitis virus (IBV) and up to 1,400 amino acids for feline coronavirus (FCoV). In
addition, this protein is highly glycosylated as it contains 21 to 35 N-glycosylation sites. Spike proteins
assemble into trimers on the virion surface to form the distinctive “corona”, or crown-like appearance.
The ectodomain of all CoV spike proteins share the same organization in two domains: a N-terminal
domain named S1 that is responsible for receptor binding and a C-terminal S2 domain responsible for
fusion (Figures 2 and 3). A notable distinction between the spike proteins of different coronaviruses is
whether it is cleaved or not during assembly and exocytosis of virions. With some exceptions, in most
alphacoronaviruses and the betacoronavirus SARS-CoV, the virions harbor a spike protein that is
uncleaved, whereas in some beta- and all gammacoronaviruses the protein is found cleaved between
the S1 and S2 domains, typically by furin, a Golgi-resident host protease (Figure 2). Interestingly,
within the betacoronavirus mouse hepatitis virus (MHV) species, different strains, such as MHV-2 and
MHV-A59 display different cleavage requirements. This has important consequences on their
fusogenicity, as detailed in Section 4. The S2 subunit is the most conserved region of the protein,
whereas the S1 subunit diverges in sequence even among species of a single coronavirus. The S1
contains two subdomains, a N-terminal domain (NTD) and a C-terminal domain (CTD). Both are able
to function as receptor binding domains (RBDs) and bind variety of proteins and sugars.
The coronavirus spike protein is a class I fusion protein [5]. The formation of an α-helical
coiled-coil structure is characteristic of this class of fusion protein, which contain in their C-terminal
part regions predicted to have an α-helical secondary structure and to form coiled-coils. Influenza
hemagglutinin protein HA is the prototypical member of the class I fusion protein family and one of
the best characterized so far [6]. HA is synthesized as a HA0 precursor and assembles into trimers. The
Viruses 2012, 4
1015
protein becomes fusion competent by cleavage of HA0 into HA1 and HA2. The fusion peptide, a very
conserved hydrophobic sequence, is located at the N-terminus of HA2. In the pre-fusion conformation,
the central coiled-coil of the trimer is formed by three long helices with three shorter helices packed
around them. In this conformation, the fusion peptide is protected, buried within the trimer interface.
Two major conformation changes occur during fusion. Upon endosomal acidification, an unstructured
linker becomes helical allowing formation of a long helix in the N-terminal part. In this conformation,
called a prehairpin, the fusion peptide is projected towards the target membrane where it is then
embedded, connecting the viral and target cell membranes. The second conformational change consists
of the inversion of the C-helix that packs into the grooves of the N-terminal trimeric coiled-coils
forming a six-helix bundle (6HB). In the resulting conformation, the transmembrane domain and the
fusion peptide anchored into the target membrane are brought in close proximity facilitating merging
of viral and cell membranes.
Coronavirus spike proteins contain two heptad repeats in their S2 domain, a feature typical of a
class I viral fusion proteins. Heptad repeats comprise a repetitive heptapeptide abcdefg with a and d
being hydrophobic residues characteristic of the formation of coiled-coil that participate in the fusion
process. For SARS-CoV and MHV, the post-fusion structures of the HR have been solved; they form
the characteristic six-helix bundle [7,8]. The functional role of MHV and SARS-CoV HR was
confirmed by mutating key residues and by inhibition experiments using HR2 peptides [9,10].
Figure 2. Severe acute respiratory syndrome (SARS)-CoV spike protein schematic. The
spike protein ectodomain consists of the S1 and S2 domains. The S1 domain contains the
receptor binding domain and is responsible for recognition and binding to the host cell
receptor. The S2 domain, responsible for fusion, contains the putative fusion peptide (blue)
and the heptad repeat HR1 (orange) and HR2 (brown). The transmembrane domain is
represented in purple. Cleavage sites are indicated with arrows.
The important role of the spike protein in cell tropism has been demonstrated with chimeric viruses.
There are many strains of mouse hepatitis virus (MHV), viruses that infect mainly the brain and liver.
Because of the different patterns of disease associated with the various strains of MHV, involvement
of their spike protein in tissue tropism has been extensively studied. The strain JHM is highly virulent
causing severe encephalitis that is often lethal, but is poorly hepatotropic. The strain MHV-A59 causes
hepatitis and mild encephalitis. MHV-2 is highly hepatotropic. By using chimeric viruses between
these different strains, it has been shown that the S protein is linked to the tropism and pathogenesis of
MHV. Introduction of JHM or MHV-2 S genes in the MHV-A59 background increases the
recombinant virus’ neurovirulence and hepatotropism respectively [11,12]. However, replacement of
JHM S protein sequence with MHV-A59 S gene in the JHM background does not confer
hepatotropism suggesting that other factors modulate virus tropism. A mutant MHV-A59 strain
exhibiting altered tropism was isolated from persistently infected microglial cells [13]. The single
Viruses 2012, 4
1016
mutation Q159L in the S1 domain is responsible for reduced replication in the liver and low
hepatotropism of the virus [14]. The important role of the spike protein in tropism has also been shown
for other coronaviruses. IBV is an important domestic fowl pathogen that replicates in the respiratory
tract but also in epithelial cells from the kidney, the oviduct and the gut. In vitro, clinical strains of
IBV infect only chicken embryo kidney cells and grow on embryonated eggs. IBV Beaudette strain is
an attenuated strain that was obtained by serial passage of IBV on eggs. IBV Beaudette, in addition to
chicken embryo kidney cells, also infects CEF, BHK-21 and Vero cells. Substitution of the S gene in
the Beaudette background with that of the IBV M41 strain restricts the tropism of the virus to primary
chicken cells [15]. However, in vivo this chimeric virus has the attenuated phenotype of Beaudette.
These data show that change in tropism of Beaudette in cell culture is mainly determined by the S
protein though the avirulence also results from attenuating mutations in other genes [16].
Feline coronaviruses (FCoV) provide a fascinating example of the critical involvement of the spike
protein in tropism and pathogenesis [17]. Within this alphacoronavirus species, there are two known
serotypes, 1 and 2, based on serological and genetic characteristics of their spike. Furthermore, there
are two biotypes within each serotype, both of which are associated with extremely contrasting
pathological potential. Cats get commonly infected with the feline enteric coronavirus (FECV), a
biotype that gives rise to usually asymptomatic to mild enteric tract infections and may establish
persistence in the host. In contrast, some FCoV-infected cats sporadically develop an invariably fatal
immune-mediated disease called feline infectious peritonitis (FIP). In this case, the causative agent is
called feline infectious peritonitis virus (FIPV). A striking characteristic of FIPVs that sets them apart
from FECVs is their ability to efficiently replicate in monocytes and macrophages [18]. It is thought
that this switch in tropism, from gut epithelium to motile monocytes/macrophages cells, is a crucial
tipping point towards the development of FIP as it allows for viral dissemination throughout the host.
The current understanding is that mutations in FECV in a persistently infected host cause it to
change into the virulent FIPV [19]. While it has been hypothesized that mutations or deletions in
certain genes, such as 3c and 7b, may be associated with the emergence of FIPV [19], the causative
mutations for the biotype switch are still unknown. There is, however, evidence that mutations in the
spike gene may play key role in the transition of tropism from gut epithelium to macrophages. Rottier
and colleagues have focused on the genetically close and laboratory-adapted type 2 FECV 79–1683
and FIPV 79–1146 pair [20]. While both viruses have similar growth characteristics in established
feline epithelial cells, only FIPV 79–1146 but not FECV 79–1683 has the ability to efficiently infect
and replicate in macrophages. Using a targeted RNA recombination system [21] the authors were able
to generate recombinant chimeric virus to determine regions of the genome that are important for
infection of bone marrow derived macrophages. They found that the exchange of the FIPV 79–1146 S
gene with that of FECV 79–1683 in the FIPV 79–1146 genetic background strongly reduced the
chimeric recombinant virus’ capacity to infect macrophages compared to the recombinant wild type
FIPV 79–1146. Furthermore, additional chimeras were generated to more precisely map the regions of
spike that are important for macrophage tropism. Surprisingly, the C-terminal region of the spike (from
residue 874 to the C-terminus) but not the N-terminal region (which contains the S1 receptor binding
domain) was found to be responsible for the macrophage tropism in this system. A total of ten amino
acid substitution differentiates the C-terminal regions of FECV 79–1683 and FIPV 79–1146, however
the precise mutation(s) that cause the tropism switch remain(s) to be determined [20].
Viruses 2012, 4
1017
While serotype 2 FCoV have been studied in a relatively detailed manner, in particular because they
propagate more easily in vitro, serotype 1 FCoV, which are more relevant clinically as they are more
prevalent, are less well understood. And although it can be assumed that viruses of both serotypes
behave in similar ways for most of their life cycle, it remains to be investigated whether the same or
different set of mutations would account for the biotype switch in the two serotypes. Thus, more
efforts are needed to study serotype 1 FCoV. Such efforts would shed light on the basis of FCoV
pathogenesis.
The difference in tropism mediated by S proteins results from different mechanisms linked to the
two main functions of the protein: receptor binding and fusion, which will be further discussed.
3. Receptor Binding and Tropism
The first coronavirus receptor identified was the MHV receptor, in 1991 [22]. MHV binds to the
adhesion molecule CEACAM1 (Carcinoembryonic antigen-cell adhesion molecule) to infect cells.
CEACAM1 is a type I transmembrane protein belonging to the immunoglobulin superfamily.
CEACAM1 is a multifunctional protein that has roles in adhesion and cell signaling, among others.
The CEACAM1 ectodomain contains four Ig constant region like domains, N, A1, B and A2. The
N-terminal domain N of CEACAM1 is involved in MHV binding [23,24]. There are two allelic forms
of CEACAM1, CEACAM1a and CEACAM1b. They can both function as receptors, however, binding
by CEACAM1a is much more efficient [23]. The involvement of receptor usage and tropism of MHV
strains have been studied. It has been shown that neurovirulence of JHM is associated with rapid
spread of the virus in the brain that is partly independent of CEACAM1. In vitro, MHV-JHM requires
CEACAM1 for entry, however, in vivo, JHM is able to infect ceacam−/− mice but with a 100-fold
higher lethal dose [25]. As a consequence, it has been suggested that JHM, in the absence of
CEACAM1, uses an alternative, less effective and yet to be determined receptor in order to initiate
infection. After primary infection, the virus could propagate very rapidly by using cell-cell fusion
independently of the receptor (receptor independent spread) [26]. In vivo, MHV-A59 is strictly
dependent on CEACAM1 for infection [27], but persistent infection of murine cells leads to the
emergence of viruses with extended tropism [28]. The MHV/BHK virus infects cells in a heparan
sulfate-dependent and CEACAM1-independent manner because of the acquisition of two heparan
sulfate binding sites in the S protein [29,30]. It has been shown that both binding sites are required to
acquire the CEACAM1-independent phenotype [30].
For JHM strain, many isolates exist that differ in their neurovirulence levels. The virulence is
correlated to the length of a hypervariable region present within S1. The isolate MHV-4 of JHM
contains the longest region and it is associated with independent CEACAM1 cell-cell fusion and
spread [31]. It has been suggested that conformational changes of the spike protein are facilitated by a
less stable association of the S1 and S2 subunits [32]. This suggests that the higher the fusogenic
potential of the spike protein is, the less the virus depends on its receptor for entry.
Among alphacoronaviruses two human viruses (HCoV-229E and HCoV-NL63) can be found along
with viruses that infect animals and can be responsible of severe illness: transmissible gastroenteritis
CoV (TGEV) and canine CoV (CCoV) cause enteric disease in pigs and dogs respectively while feline
coronaviruses cause enteric and systemic disease in cats.
Viruses 2012, 4
1018
HCoV-229E, TGEV, serotype 2 FCoV and CCoV all use the aminopeptidase N (APN) protein of
their natural host as receptor. Interestingly, in addition to their specific host APN, these viruses are
able to bind the feline APN. It has been suggested that these viruses may have originated from a
common ancestor coronavirus infecting felines that used APN as a receptor [2]. APN, also known as
CD13, is a type II transmembrane protein expressed on the apical domain of epithelial cells of
respiratory and enteric tracts. APN is a Zn2+ dependent protease that preferentially degrades peptides
or proteins with a N-terminal neutral amino acid. It has been shown that tropism differences of these
viruses are due to the ability of their spike proteins to recognize small species-specific amino acid
differences in APN [33]. Spike proteins of HCoV-229E, TGEV, FCoV and CCoV present a high
homology, however, binding domains are located in non-homologous regions.
TGEV infects epithelial cells from the small intestine but is also able to infect cells from the
respiratory tract. In the mid-1980s, an attenuated variant of TGEV, porcine respiratory coronavirus
(PRCoV) was isolated in Belgium. This virus provides an example of altered tissue tropism due to a
deletion occurring in the spike gene [34]. Unlike TGEV, PRCoV infects only pulmonary epithelial
cells. Both spike proteins bind porcine APN, the receptor binding domain being located between
residues 522 and 744 of TGEV S protein. The spike protein of TGEV has a hemagglutinating activity
that is absent in PRCoV as this activity is contained in the deleted N-terminal part of the protein [35].
One of the consequences of this lack of activity is the inability of PRCoV to replicate in the gut. The
hemagglutinating activity was mapped to the residues 145–155 of TGEV spike protein and it has been
shown that a mutation abrogating this activity reduced the enteropathogenicity of the virus [36]. In
addition, a study has shown that the sialic acid binding activity of TGEV is responsible for binding of
an additional protein designated as mucin-like glycoprotein (MPG) in brush border membranes [37]. It
has been suggested that this binding might shield the virus from the action of gut emulsifiers [38]. The
role of the NTD and carbohydrate binding in TGEV provides interesting insights into coronavirus
enterotropism, a property that generally is attributed to non-enveloped viruses. The role of the NTD in
other enteric alphacoronaviruses such as FCoV and canine coronavirus (CCoV) is still unknown.
Other coronaviruses have sialic acid binding activity, in particular bovine coronavirus (BCoV) and
human HCoV-OC43 [39]. The ability of betacoronaviruses to bind carbohydrates has been mapped to
a galectin fold-like structure present in the S1 NTD [40]. So far, besides the binding of Neu5,9Ac2
conjugates, no other specific receptors have been identified for these viruses. They belong to the
betacoronavirus group and contain HE proteins, so they resemble influenza virus as they have a
receptor-destroying enzyme. However, the exact role of HE during coronavirus entry remains unclear.
IBV also exhibits sialic acid binding activity but the role of such activity in pathogenicity is not
known. For IBV, extended host range of Beaudette strains in cell culture has been linked to the
presence of a heparin binding site in the spike protein [41].
Another example of heparan sulfate binding is found with type 1 FCoV spike. By incubating viruses
with heparin-agarose beads (heparin has a very similar structure to heparan sulfate, and is used in
binding assays) de Haan and colleagues have demonstrated by quantification of bead-associated viral
RNA that the cell-culture-adapted type 1 FIPV UCD1 strain can bind heparin [42]. Very interestingly,
the putative heparin binding motif proposed by the authors resides in a defective furin cleavage site at
the boundary between the S1 and S2 domains. The type 2 FIPV 79–1146 as well as the UCD1-related
type 1 FIPV UCD, which harbors a functional furin cleavage site, were not able to bind the heparin
Viruses 2012, 4
1019
beads. This lends support to the notion that an uncleaved heparan sulfate recognition motif is required
for binding activity. Furthermore, the authors found that inoculation of UCD1 to FCWF cells in the
presence of competing heparin severely diminished infection. Infection by type 2 FIPV 79–1146 was
not affected by this heparin competition assay.
As mentioned above, the coronavirus spike protein/receptor pairing is a key determinant of tropism.
To infect a new host species, coronaviruses must adapt to the receptor of their new host either by
mutation or by recombination with a coronavirus infecting their new host. In the case of SARS-CoV,
the virus appeared in 2002 in live animal retail markets in China. Related viruses were isolated from
Himalayan palm civets, raccoon dogs and Chinese ferrets; however, it is believed that these animals
were not the reservoir of the virus, but intermediate hosts during the species-jumping event. The
receptor of the SARS-CoV is the angiotensin-converting enzyme 2 (ACE2) [43]. ACE2 is a type I
integral membrane protein abundantly expressed in lung tissue; it is a mono-carboxypeptidase that
hydrolyses angiotensin II. Human and Himalayan palm civet coronavirus receptor usage analyses have
shown that human SARS-CoV can bind both human and palm civet ACE2 whereas the palm civet
virus cannot bind hACE2. It has been shown that adaptation of the virus to humans was due to two
point mutations, K479N and S487T, in the binding domain of the SARS-CoV S protein [44]. Further
characterization by Wu et al. of adaptive mutations of the RBD led to the identification of mutations
that strengthen the interaction with either human or palm civet ACE2 [45]. SARS-CoV-like viruses
have been isolated in bats. In this case, entry does not occur via ACE2 and their receptor(s) is/are
unknown; however, replacement of the amino acid sequence found between residues 323 and 505 with
the corresponding sequence of the SARS-CoV RBD is sufficient to allow human ACE2 receptor
usage [46].
Coronaviruses are able to exploit many cell surface molecules—proteins and carbohydrates alike—
in order to gain entry into target cells. Host calcium dependent (C-type) lectins have been recognized
to play a role in infection by SARS-CoV, IBV, and FCoV. Dendritic cell-specific intercellular
adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a C-type lectin expressed on macrophages
and dendritic cells. Its function is to recognize high mannose glycosylation patterns commonly found
on viral and bacterial pathogens. Viral exploitation of DC-SIGN is best documented in HIV-1, which
attaches via N-glycosylated residues on the surface of the virus. HIV-1 uses DC-SIGN to subvert the
host immune defenses by entering and initiating infection of dendritic cells or macrophages directly
(in-cis), or by traveling with the cell to lymph nodes where the virus is transferred to T-cells at the
immunological synapse (in-trans). Like HIV-1 gp120, the coronavirus spike is heavily glycosylated
providing the virus with the opportunity to interact with host lectins such as DC/L-SIGN. L-SIGN,
which is expressed on endothelial cells of the liver as well as in the lung, has been reported to be an
alternate receptor for SARS-CoV and HCoV-229E [47,48]. In-trans transmission of SARS-CoV by
dendritic cells to susceptible target cells has been documented. Although the dendritic cells studied
were capable of transferring infectious virions via a synapse-like structure, in-cis infection was not
observed [49]. Site directed mutagenesis has identified glycosylation at the asparagine residues 109,
118, 119, 158, 227, 589, and 699 as critical for L-SIGN/DC-SIGN mediated entry [50].
FIPV is an example of a coronavirus that targets immune cells—specifically monocytes and
macrophages—to achieve systemic spread. Infection of non-permissive cell types was achieved
through exogenous expression of DC-SIGN, demonstrating that both type 1 and type 2 FIPVs use
Viruses 2012, 4
1020
DC-SIGN as a co-receptor or as an alternative receptor to fAPN, respectively [51,52]. In the case of
IBV, experiments have demonstrated that DC-SIGN and the closely related L-SIGN enhance infection
of otherwise non-permissive cells, in a sialic acid-independent fashion [53]. The role of lectins in IBV
infection in vivo is undetermined.
4. Entry and Fusion
Enveloped virus entry can occur directly at the cell surface after binding to the receptor or after
internalization via endocytosis with fusion taking place in the endosomal compartment. Fusion of viral
membranes with host membranes is driven by large conformational changes of the spike protein. Over
time, coronaviruses have modified their spike proteins, leading to the diversity of triggers used to
activate their fusion. These conformational changes can be initiated by receptor binding but may need
additional triggers such as pH acidification or proteolytic activation. The mechanisms of coronavirus
entry are complex and differ between coronavirus species and strains. For example, depending on the
MHV strain, fusion can occur directly at the cell surface after receptor binding or after endocytosis.
JHM strain MHV-4 fuses at neutral pH, but the virus was also detected in endosomal vesicles [54]. It
is likely that MHV-4 is capable of entering directly at the cell surface or through the endosomal
pathway. The choice of entry mechanism may depend on the cell type. In both cases, fusion is solely
triggered by receptor binding. Indeed, it has been shown that incubation of JHM spike protein with a
soluble form of the receptor CEACAM1 induces modifications in S hydrophobicity and
conformational change of the S2 region [55–57]. In addition, JHM is able to spread from DBT cells to
BHK cells that do not express the MHV receptor. Incubation with soluble receptor increases this
receptor-independent propagation [58]. The capacity of the spike protein to fuse at neutral pH relies on
the properties of the fusion machinery. A variant of MHV-4 isolated from persistently infected cells
requires low pH exposure for productive infection. The difference of pH requirement for fusion
between the mutant and the wild type virus was attributed to three point mutations in the heptad repeat
regions [59]. Concerning MHV-A59 entry mechanisms, contradictory results have been reported.
Qui et al. reported that parental strain of MHV-A59 was insensitive to lysomotropic agent whereas the
recombinant strain containing the MHV-2 spike protein relies on low pH for entry [60]. Indeed,
MHV-2 entry requires low-pH-activated endosomal proteases (cathepsin B and L) and, if a cleavage
site is introduced in MHV-2 spike protein, virus entry no longer requires these proteases; these data are
in favor of pH-independent fusion induced by receptor binding. Eifart et al. challenged this scenario.
Combining different approaches of infection and microscopy, the authors have shown that MHV-A59
infection is sensitive to lysomotropic agent and that the virus is internalized allowing initiation of
infection, suggesting that pH acidification is required to trigger viral fusion [61]. It is likely that
receptor binding is a key determinant of MHV entry, however the requirement for an additional fusion
trigger remains unclear. For MHV-2, the endocytosis mechanism was further characterized: the virus is
internalized by a clathrin-dependent pathway that does not depend on the eps15 adaptor [62].
Endosomal pH acidification is a fusion trigger for many viruses such as influenza virus and
vesicular stomatitis virus (VSV). For many years, it was believed that IBV fusion occurs at neutral pH
as infected cells form large syncytia at neutral pH. However, it has been shown by Chu et al. that
infection is blocked by lysomotropic agent and that IBV fusion process was activated by low pH [63].
Viruses 2012, 4
1021
The authors directly assessed the pH dependence of IBV fusion in fluorescence dequenching assays.
They showed that fusion occurs at acidic pH, with a half-maximal fusion rate occurring at pH 5.5. In
order to infect cells, IBV enters by endocytosis. Inhibitory drugs of the clathrin-mediated pathway
such as chlorpromazine also abolished IBV infection. IBV virions harbor cleaved spike proteins, with
the cleavage occurring between the S1 and S2 domains. The IBV Beaudette strain has the peculiar
feature to contain a second furin cleavage site in the S2 domain of the spike. Infection and syncytia
formation is inhibited by the presence of a furin inhibitor. Mutation or deletion of the S1–S2 cleavage
site in Beaudette spike protein delays virus propagation but does not abolish syncytia formation. These
mutants are still sensitive to furin inhibition. Mutants containing a minimal cleavage site XXXR690/S
are infectious, however they are dependent on serine proteases for productive infection [64].
The relationship between the cleavability of the coronavirus spike protein and its fusogenicity has
been a source of debate for researchers for many years. When viral fusion proteins are expressed at the
cell surface, activation of viral fusion results in cell-cell fusion and formation of giant multinucleated
cells named syncytia. It is generally believed that syncytia formation, which involves cell membranes
fusing together, reflects the fusion process between viral and host cell membranes. However, it now
appears that cell-cell and virus-cell fusion mechanisms may differ. Indeed, because of differences in
factors such as membrane curvature and/or density of viral envelope glycoproteins, the processes
involved in cell-cell and virus-cell fusion may considerably differ mechanistically. Furthermore,
formation of syncytia in infected cells is not observed with all coronaviruses. Cleavage of the fusion
protein is a common characteristic of class I viral fusion proteins. For influenza, the nature of the
cleavage site of HA is of great importance in virus pathogenicity. The cleavage event required to prime
the protein for fusion can occur in the secretion pathway by furin or during infection by host proteases
of the respiratory tract. Influenza virus strains that are cleaved by host furin are highly pathogenic as
they cause systemic infection [65]. For coronaviruses, the relationship between cleavage of the S
protein and its cell-cell fusion capability has been well established. Cleaved proteins show a higher
propensity for cell-cell fusion. Introduction of a mutation H716D in the spike protein of MHV-A59
strongly impaired the cleavage of the protein and delayed cell-cell fusion [66]. MHV-2 strain spike
protein is not cleaved and mutation of the sequence of MHV-A59 cleavage site with the corresponding
sequence of MHV-2 S protein delays cell-cell fusion. Introduction of a cleavage site in MHV-2 spike
protein induces the formation of syncytia at neutral pH. In vitro spike protein cleavage plays an
important role in fusogenicity, however the role in virus-fusion and pathogenicity is less clear. For
example, MHV-A59 bearing the mutation H716D is very similar to the wild type virus in terms of
pathogenicity [67]. MHV-A59 viral stock produced in presence of a furin inhibitor to block spike
cleavage enters cells with kinetics similar to the wild type virus [68]. Moreover, a study by Hingley
and collaborators has shown that the spike of virus purified from liver homogenates of MHV-A59-
infected mice was not cleaved, further adding evidence that proteolytic processing of spike is not
essential for entry and spread in vivo [69]. For coronaviruses that have a spike that is cleaved by furin,
it is important to note that this protease belongs to a family of enzymes called proprotein convertases
(PCs), which has nine members [70]. Along with furin, six other PCs proteases, PC1, PC2, PC4, PC5,
PACE4 and PC7, share the same basic recognition motif: R/K-[X]0,2,4,6-R/K (where X is any amino
acid) [71]. It remains to be determined whether other PCs that are related to furin can also recognize
and cleave coronavirus spike proteins.
Viruses 2012, 4
1022
For some coronaviruses that harbor a non-cleaved spike protein on their surface, such as MHV-2
and SARS-CoV, it has been shown that they rely on endosomal proteases for productive entry. Indeed,
MHV-2 entry depends on host cell cathepsin L and B [60]. This dependence is abolished by the
introduction of a furin cleavage site between the S1 and S2 domains. For SARS-CoV, the link between
cleavage and fusion is more complex (Table 2). It has been shown that SARS-CoV infection is
inhibited by lysomotropic agents because of the inhibition of the low-pH-actived protease cathepsin
L [72]. In addition cell-cell and virus-cell fusion can be triggered by trypsin treatment [73]. This led to
the hypothesis that SARS-CoV fusion was triggered by proteolytic processing of the spike protein. It
has been shown that different proteases enhance SARS-CoV infection in vitro: trypsin, thermolysin
and elastase [74]. Analysis of SARS-CoV spike protein processing by trypsin and elastase had shed
light on SARS-CoV fusion [75,76]. It was shown that trypsin activates fusion by sequential cleavage
of the spike protein at two discrete sites. The first cleavage event at the S1–S2 boundary (R667)
probably facilitates the second cleavage event at the position R797 (S2’ region) that is responsible for
fusion activation [75,77]. The second cleavage occurs directly at the N-terminal extremity of the fusion
peptide. Cleavage at R667 is dispensable for fusion activation but enhances cell-cell or virus-cell
fusion. Elastase mediates cleavage at the residue T795, not directly next to the fusion peptide.
SARS-CoV spike protein shows a certain degree of plasticity in the position of the cleavage site for
priming of fusion. Conversely, when influenza HA is cleaved by Pseudomonas elastase the cleavage
position is shifted by one amino acid, which leads to fusion incompetency [78]. SARS-CoV S
residue 795 is probably less accessible for cleavage than residue 797 and the fusion induced by
cleavage at position 797 is more efficient. The difference of fusion efficacy may also result from its
location at the N-terminus of the fusion peptide. These data suggest that fusion is modulated by spatial
regulation of the cleavage site. It has been shown that cathepsin L cleaves the SARS-CoV spike
protein in the S1–S2 boundary region at residue T678, however, so far, cleavage in the S2’ region has
yet to be conclusively demonstrated [79].
SARS-CoV is able to fuse directly at the cell surface in the presence of relevant exogenous
protease. It is believed that this route of entry is 100- to 1000-fold more efficient than the endosomal
pathway [74]. The availability of proteases in the extracellular milieu is a key factor of tropism.
SARS-CoV is a respiratory pathogen and it has been known for a long time that proteases from the
respiratory tract such as members of the transmembrane protease/serine subfamily (TMPRSS),
TMPRSS2 or HAT (TMPRSS11d) are able to cleave influenza HA [80]. Indeed, TMPRSS2 and HAT
(or TMPRSS11d) are both able to induce SARS-CoV fusion [81–84]. Infection of target cells with
SARS-CoV S-pseudotyped virions is less sensitive to cathepsin inhibitors when target cells express
TMPRSS2 [83,84]. SARS-CoV S-pseudotyped virions produced in cells expressing TMPRSS2 still
rely on endosomal cathepsin for entry, while they are less sensitive to neutralizing antibodies. This
effect was attributed to the release of spike fragments in the supernatant that lure antibodies and may
be of great importance for the spread of the virus [83]. It has been shown that processing of the spike
protein by HAT and TMPRSS2 may differ: HAT cleaves the SARS-CoV S protein mainly at R667
whereas TMPRSS2 cleaves the protein at multiple sites, notably in a region near S2’, although the
precise locations of the cleavage sites for this protease remain to be determined [81]. Expression of
HAT in target cells does not confer NH4Cl or cathepsin inhibitor resistance to SARS-CoV
S-pseudotyped virion entry [84]. It is likely that spatial and temporal modulation of activation by
Viruses 2012, 4
1023
proteases play important roles. Cleavage of incoming virions before their binding to the cell would
probably abort infection by inactivating the virion. Interestingly, TMPRSS2 is associated with ACE2,
the SARS-CoV receptor [81]. TMPRSS2 likely plays a key role in the initial infection and spread of
the virus; however the importance of this protease on SARS-CoV infection results from a fine balance
between two antagonist effects on infection: shedding of the receptor and fusion activation.
Table 2. Proteolytic processing of the SARS-CoV spike protein important for cell-cell
fusion and/or virus entry. The table summarizes the different proteases involved in the
cleavage of SARS-CoV spike protein and their roles in activating cell-cell fusion and/or
virus entry.
Protease Cleavage site Cell-cell fusion
Virus Entry
Cathepsin L [72,79]
S1/S2 HTVSLLRSTSQKSIVAYTMSL - +
Elastase [74,85] S2’ LPDPLKPTKRSFIEDLLFNKV + +
HAT (TMPRSS11d)
[84] S1/S2 HTVSLLRSTSQKSIVAYTMSL + -
Plasmin [82] S1/S2 HTVSLLRSTSQKSIVAYTMSL
S2’ LPDPLKPTKRSFIEDLLFNKV n.d. +
TMPRSS11a [82] S1/S2 HTVSLLRSTSQKSIVAYTMSL
S2’ LPDPLKPTKRSFIEDLLFNKV n.d. +
TMPRSS2 [81,83,84]
Multiple sites + +
Trypsin [73,75] S1/S2 HTVSLLRSTSQKSIVAYTMSL
S2’ LPDPLKPTKRSFIEDLLFNKV + +
For FCoV, a well-studied case for the role of proteolytic activation of spike comes from research on
the type 2 FCoV pair FECV 79-1683 and FIPV 79–1146 [86]. By using specific cathepsin inhibitors,
the authors have shown that the two strains differ substantially in their use of activating proteases used
during entry. While the FECV strain 79–1683 was found to rely on both cathepsin B and L as well as
on an acidic endosomal environment, the FIPV strain 79–1146 was dependent on cathepsin B activity
only. This was further confirmed by a biochemical assay that found that FECV 79–1683 can be
cleaved by cathepsin B and L, whereas FIPV 79–1146 could only be cleaved by cathepsin B. Based on
the molecular weights of the cathepsin cleavage products, it was hypothesized that the cleavage site
did not reside in the boundary region between the S1 and S2 domain, but in a region located in the
C-terminal part of S2 [86].
5. Fusion Peptide
A critical feature of any viral fusion protein is the so-called “fusion peptide”, which is a relatively
apolar region of 15–25 amino acids that interacts with membranes and plays an essential role in the
fusion reaction [6,87,88]. Fusion peptides of class I viral fusion proteins are typically classified as
“external” or “internal” depending on their location relative to the proteolytic cleavage site [89]. One
Viruses 2012, 4
1024
key feature of viral fusion peptides is that within a particular virus family, there is high conservation of
amino acid residues; however, there is little similarity between fusion peptides of different virus
families [90]. In the case of influenza HA, which is a classic example of an “external” fusion peptide,
the N- and C-terminal parts of the fusion peptide (which are α-helical) penetrate the outer leaflet of the
target membrane, with a kink at the phospholipid surface. The inside of the kink contains hydrophobic
amino acids, with charged residues on the outer face [91]. Internal fusion peptides (such as the one
found in the Ebola virus GP) often consist of loops, but also require a mixture of hydrophobic and
flexible residues similar to N-terminal fusion peptides [87,92]. It is important to note that despite the
presence of key hydrophobic residues, viral fusion peptides often do not display extensive stretches of
hydrophobicity, and can contain one or more charged residues [93].
To date, the exact location and sequence of the coronavirus fusion peptide is not known [94],
however, by analogy with other class I viral fusion proteins, it is predicted to be in the S2 domain. The
location of the fusion peptide has been most extensively studied for SARS-CoV. Three membranotropic
regions in SARS-CoV S2 were originally suggested as potential fusion peptides [95,96]. Based on
sequence analysis and a hydrophobicity analysis of the S protein using the Wimley-White (WW)
interfacial hydrophobic interface scale, initial indications were that the SARS-CoV fusion peptide
resided in the N-terminal part of HR1 [5,97], which is conserved across the Coronaviridae. Mutagenesis
of this predicted fusion peptide inhibited fusion in syncytia assays of S-expressing cells [98]. This
region of SARS-CoV has also been analyzed by other groups in biochemical assays [96,99,100] and
was defined as the WW II region (residues 864–886)—although Sainz et al. [99] actually identified
another, less conserved and less hydrophobic, region (WW I, residues 770–778) as being most
important for fusion. Peptides corresponding to this region have also been studied in biochemical
assays by other groups [101]. In addition, a third, aromatic region adjacent to the transmembrane domain
(the membrane-proximal domain) has been shown to be important in SARS-CoV fusion [102–105]. This
membrane-proximal domain likely acts in concert with a fusion peptide in the S2 ectodomain to
mediate final bilayer fusion once conformational changes have exposed the fusion peptide in the
ectodomain.
Based on the finding that SARS-CoV S can be proteolytically cleaved at a downstream position in
S2, at residue 797 [75–77], further investigations were carried out to determine whether cleavage at
this internal position in S2 might expose a domain with properties of a viral fusion peptide. A
mutagenesis study of SARS-CoV S residues 798–815, a stretch located in between the WW I and WW
II regions, combined with lipid-mixing and structural studies of an isolated peptide, showed the
importance of this region as a novel fusion peptide for SARS-CoV. The sequence immediately
C-terminal to the R797 cleavage site of SARS-CoV S is SFIEDLLFNKVTLADAGF, and it is notable
that both R797 and this downstream sequence are highly conserved across the Coronaviridae.
In particular, the IEDLLF motif showed only minimal divergence, with occasional conservative
substitutions [106].
Examination of the proposed IEDLLF fusion peptide in the context of a structural model of the
SARS-CoV S homotrimer shows that it is externally positioned mid-way down the trimer, and as such,
would appear to be appropriately located to function as a fusion peptide. Notably, L803, L804 and
F805 are the initial residues of a major antigenic determinant of SARS-CoV S (Leu 803–Ala 828) that
is capable of inducing neutralizing antibodies [107]. This SARS-CoV epitope is also homologous to an
Viruses 2012, 4
1025
immunodominant neutralizing domain (the 5B19 epitope) on the MHV S2 subunit [108]. While a
crystal structure of the SARS-CoV S ectodomain has not yet be solved, a predictive model of the
quaternary structure is available: PDB entry 1T7G [109]. In the context of this model, the novel S2
fusion peptide is mainly helical (especially the conserved residues SxIEDLLF), with a short central
unstructured region, and is in a relatively exposed position mid-way down the trimeric spike protein
complex (Figure 3). This structure and position within the S trimer is consistent with its function as a
viral fusion peptide. Comparisons with other fusion proteins reveal some similarities to the “internal”
fusion peptides of Ebola virus and avian leukosis virus. Like these viruses, the coronavirus fusion
peptide is exposed by proteolytic cleavage [6,89,110], yet is not a classic “external” fusion peptide like
influenza HA.
Figure 3. SARS-CoV spike protein three-dimensional predicted structure. This representation,
shows the trimeric (inset) and monomeric forms of the protein and is based on the
three-dimensional predicted model found in the PDB database (PDB entry 1T7G, [109]).
Note that the predicted model does not include residues 681-736 of the protein. The S1 and
S2 domains as well as the cleavage sites and putative fusion peptide are highlighted.
6. Conclusions
In the past ten years, many new coronaviruses have been identified. They infect a wide range of
hosts from mammals to birds. Closely related coronaviruses have been identified in distantly related
animals suggesting recent interspecies jumps. Coronavirus diversity is fundamentally due to the low
fidelity of the virally encoded RNA-dependent-RNA-polymerase that generates around 10−3 to 10−5
substitutions per site per year. The large size and replication strategy of the coronavirus genome also
allows for frequent homologous recombination, a process that enables exchange of genetic material
during co-infection. Persistent infection leads to the accumulation of adaptive mutations. The
consequences of coronavirus species barrier jumping can be devastating and result in severe disease
and mortality, as exemplified by the SARS outbreak. The spike protein is the major determinant of
Viruses 2012, 4
1026
coronaviruses tropism. Modification of the spike can alter cell and tissue tropism and, in some cases, in
association with other viral and host factors, may lead to change of virus pathogenicity. Zoonoses
constitute a real risk for human health. In the past, coronaviruses have often demonstrated their
propensity to infect new hosts, highlighting the capacity for viral evolution and the need for
surveillance. Great progress has been made in the understanding of spike protein functions, however it
remains impossible to predict the effect of mutations that a virus might acquire.
Acknowledgments
We apologize to all authors whose work could not be cited due to space restrictions. We thank
Sophana Ung (Center for Infection & Immunity of Lille) for preparing the illustrations. Work in the
author’s lab (G.W) is supported by research grants from the National Institutes of Health (R21
AI1076258), the Winn Feline Foundation, the Morris Animal Foundation, and the Cornell Feline
Health Center.
Conflict of Interest
The authors declare no conflict of interest.
References and Notes
1. Hudson, C.B.; Beaudette, F.R. Infection of the cloaca with the virus of infectious bronchitis.
Science 1932, 76, 34.
2. Weiss, S.R.; Navas-Martin, S. Coronavirus pathogenesis and the emerging pathogen severe acute
respiratory syndrome coronavirus. Microbiol. Mol. Biol. Rev. 2005, 69, 635–664.
3. Perlman, S.; Netland, J. Coronaviruses post-SARS: Update on replication and pathogenesis.
Nat. Rev. Microbiol. 2009, 7, 439–450.
4. Enjuanes, L.; Almazan, F.; Sola, I.; Zuniga, S. Biochemical aspects of coronavirus replication and
virus-host interaction. Annu. Rev. Microbiol. 2006, 60, 211–230.
5. Bosch, B.J.; van der Zee, R.; de Haan, C.A.; Rottier, P.J. The coronavirus spike protein is a class I
virus fusion protein: Structural and functional characterization of the fusion core complex.
J. Virol. 2003, 77, 8801–8811.
6. White, J.M.; Delos, S.E.; Brecher, M.; Schornberg, K. Structures and mechanisms of viral
membrane fusion proteins: Multiple variations on a common theme. Crit. Rev. Biochem. Mol.
Biol. 2008, 43, 189–219.
7. Xu, Y.; Cole, D.K.; Lou, Z.; Liu, Y.; Qin, L.; Li, X.; Bai, Z.; Yuan, F.; Rao, Z.; Gao, G.F.
Construct design, biophysical, and biochemical characterization of the fusion core from mouse
hepatitis virus (a coronavirus) spike protein. Protein Expr. Purif. 2004, 38, 116–122.
8. Supekar, V.M.; Bruckmann, C.; Ingallinella, P.; Bianchi, E.; Pessi, A.; Carfi, A. Structure of a
proteolytically resistant core from the severe acute respiratory syndrome coronavirus S2 fusion
protein. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 17958–17963.
Viruses 2012, 4
1027
9. Chan, W.E.; Chuang, C.K.; Yeh, S.H.; Chang, M.S.; Chen, S.S. Functional characterization of
heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome
coronavirus. J. Virol. 2006, 80, 3225–3237.
10. Luo, Z.; Matthews, A.M.; Weiss, S.R. Amino acid substitutions within the leucine zipper domain
of the murine coronavirus spike protein cause defects in oligomerization and the ability to induce
cell-to-cell fusion. J. Virol. 1999, 73, 8152–8159.
11. Navas, S.; Seo, S.H.; Chua, M.M.; Das Sarma, J.; Lavi, E.; Hingley, S.T.; Weiss, S.R. Murine
coronavirus spike protein determines the ability of the virus to replicate in the liver and cause
hepatitis. J. Virol. 2001, 75, 2452–2457.
12. Navas, S.; Weiss, S.R. Murine coronavirus-induced hepatitis: JHM genetic background eliminates
A59 spike-determined hepatotropism. J. Virol. 2003, 77, 4972–4978.
13. Hingley, S.T.; Gombold, J.L.; Lavi, E.; Weiss, S.R. MHV-A59 fusion mutants are attenuated and
display altered hepatotropism. Virology 1994, 200, 1–10.
14. Leparc-Goffart, I.; Hingley, S.T.; Chua, M.M.; Phillips, J.; Lavi, E.; Weiss, S.R. Targeted
recombination within the spike gene of murine coronavirus mouse hepatitis virus-A59: Q159 is a
determinant of hepatotropism. J. Virol. 1998, 72, 9628–9636.
15. Casais, R.; Dove, B.; Cavanagh, D.; Britton, P. Recombinant avian infectious bronchitis virus
expressing a heterologous spike gene demonstrates that the spike protein is a determinant of cell
tropism. J. Virol. 2003, 77, 9084–9089.
16. Hodgson, T.; Casais, R.; Dove, B.; Britton, P.; Cavanagh, D. Recombinant infectious bronchitis
coronavirus Beaudette with the spike protein gene of the pathogenic M41 strain remains
attenuated but induces protective immunity. J. Virol. 2004, 78, 13804–13811.
17. Haijema, B.J.; Rottier, P.; de Groot, R.P. Feline coronaviruses: A tale of two-faced types. In
Coronaviruses Molecular and Cellular Biology; Thiel, V., Ed.; Caister Academic Press: Norfolk,
UK, 2007; pp. 183–204.
18. Pedersen, N.C. Virologic and immunologic aspects of feline infectious peritonitis virus infection.
Adv. Exp. Med. Biol. 1987, 218, 529–550.
19. Vennema, H.; Poland, A.; Foley, J.; Pedersen, N.C. Feline infectious peritonitis viruses arise by
mutation from endemic feline enteric coronaviruses. Virology 1998, 243, 150–157.
20. Rottier, P.J.M.; Nakamura, K.; Schellen, P.; Volders, H.; Haijema, B.J. Acquisition of
macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by
mutations in the feline coronavirus spike protein. J. Virol. 2005, 79, 14122–14130.
21. Haijema, B.J.; Volders, H.; Rottier, P.J. Switching species tropism: An effective way to
manipulate the feline coronavirus genome. J. Virol. 2003, 77, 4528–4538.
22. Dveksler, G.S.; Pensiero, M.N.; Cardellichio, C.B.; Williams, R.K.; Jiang, G.S.; Holmes, K.V.;
Dieffenbach, C.W. Cloning of the mouse hepatitis virus (MHV) receptor: Expression in human
and hamster cell lines confers susceptibility to MHV. J. Virol. 1991, 65, 6881–6891.
23. Taguchi, F.; Hirai-Yuki, A. Mouse hepatitis virus receptor as a determinant of the mouse
susceptibility to MHV infection. Front. Microbiol. 2012, 3, 68.
Viruses 2012, 4
1028
24. Dveksler, G.S.; Pensiero, M.N.; Dieffenbach, C.W.; Cardellichio, C.B.; Basile, A.A.; Elia, P.E.;
Holmes, K.V. Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody
bind to the N-terminal domain of cellular receptor. Proc. Natl. Acad. Sci. U. S. A. 1993, 90,
1716–1720.
25. Miura, T.A.; Travanty, E.A.; Oko, L.; Bielefeldt-Ohmann, H.; Weiss, S.R.; Beauchemin, N.;
Holmes, K.V. The spike glycoprotein of murine coronavirus MHV-JHM mediates receptor-
independent infection and spread in the central nervous systems of Ceacam1a-/- Mice. J. Virol.
2008, 82, 755–763.
26. Nakagaki, K.; Taguchi, F. Receptor-independent spread of a highly neurotropic murine
coronavirus JHMV strain from initially infected microglial cells in mixed neural cultures. J. Virol.
2005, 79, 6102–6110.
27. Hemmila, E.; Turbide, C.; Olson, M.; Jothy, S.; Holmes, K.V.; Beauchemin, N. Ceacam1a-/- mice
are completely resistant to infection by murine coronavirus mouse hepatitis virus A59. J. Virol.
2004, 78, 10156–10165.
28. Schickli, J.H.; Zelus, B.D.; Wentworth, D.E.; Sawicki, S.G.; Holmes, K.V. The murine
coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an
extended host range. J. Virol. 1997, 71, 9499–9507.
29. de Haan, C.A.; Te Lintelo, E.; Li, Z.; Raaben, M.; Wurdinger, T.; Bosch, B.J.; Rottier, P.J.
Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in
receptor binding and extended host range. J. Virol. 2006, 80, 10909–10918.
30. de Haan, C.A.; Li, Z.; te Lintelo, E.; Bosch, B.J.; Haijema, B.J.; Rottier, P.J. Murine coronavirus
with an extended host range uses heparan sulfate as an entry receptor. J. Virol. 2005, 79,
14451–14456.
31. Phillips, J.J.; Chua, M.; Seo, S.H.; Weiss, S.R. Multiple regions of the murine coronavirus spike
glycoprotein influence neurovirulence. J. Neurovirol. 2001, 7, 421–431.
32. Krueger, D.K.; Kelly, S.M.; Lewicki, D.N.; Ruffolo, R.; Gallagher, T.M. Variations in disparate
regions of the murine coronavirus spike protein impact the initiation of membrane fusion. J. Virol.
2001, 75, 2792–2802.
33. Tusell, S.M.; Schittone, S.A.; Holmes, K.V. Mutational analysis of aminopeptidase N, a receptor
for several group 1 coronaviruses, identifies key determinants of viral host range. J. Virol. 2007,
81, 1261–1273.
34. Rasschaert, D.; Duarte, M.; Laude, H. Porcine respiratory coronavirus differs from transmissible
gastroenteritis virus by a few genomic deletions. J. Gen. Virol. 1990, 71, 2599–2607.
35. Schultze, B.; Krempl, C.; Ballesteros, M.L.; Shaw, L.; Schauer, R.; Enjuanes, L.; Herrler, G.
Transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has
a sialic acid (N-glycolylneuraminic acid) binding activity. J. Virol. 1996, 70, 5634–5637.
36. Krempl, C.; Schultze, B.; Laude, H.; Herrler, G. Point mutations in the S protein connect the sialic
acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.
J. Virol. 1997, 71, 3285–3287.
37. Schwegmann-Wessels, C.; Zimmer, G.; Schroder, B.; Breves, G.; Herrler, G. Binding of
transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins. J. Virol.
2003, 77, 11846–11848.
Viruses 2012, 4
1029
38. Krempl, C.; Ballesteros, M.L.; Zimmer, G.; Enjuanes, L.; Klenk, H.D.; Herrler, G.
Characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by
analysis of haemagglutination-deficient mutants. J. Gen. Virol. 2000, 81, 489–496.
39. Schwegmann-Wessels, C.; Herrler, G. Sialic acids as receptor determinants for coronaviruses.
Glycoconj. J. 2006, 23, 51–58.
40. Peng, G.; Sun, D.; Rajashankar, K.R.; Qian, Z.; Holmes, K.V.; Li, F. Crystal structure of mouse
coronavirus receptor-binding domain complexed with its murine receptor. Proc. Natl. Acad. Sci.
U. S. A. 2011, 108, 10696–10701.
41. Madu, I.G.; Chu, V.C.; Lee, H.; Regan, A.D.; Bauman, B.E.; Whittaker, G.R. Heparan sulfate is a
selective attachment factor for the avian coronavirus infectious bronchitis virus Beaudette.
Avian. Dis. 2007, 51, 45–51.
42. de Haan, C.A.M.; Haijema, B.J.; Schellen, P.; Schreur, P.W.; te Lintelo, E.; Vennema, H.; Rottier,
P.J.M. Cleavage of group 1 coronavirus spike proteins: How furin cleavage is traded off against
heparan sulfate binding upon cell culture adaptation. J. Virol. 2008, 82, 6078–6083.
43. Li, W.; Moore, M.J.; Vasilieva, N.; Sui, J.; Wong, S.K.; Berne, M.A.; Somasundaran, M.;
Sullivan, J.L.; Luzuriaga, K.; Greenough, T.C.; et al. Angiotensin-converting enzyme 2 is a
functional receptor for the SARS coronavirus. Nature 2003, 426, 450–454.
44. Li, W.; Zhang, C.; Sui, J.; Kuhn, J.H.; Moore, M.J.; Luo, S.; Wong, S.K.; Huang, I.C.; Xu, K.;
Vasilieva, N.; et al. Receptor and viral determinants of SARS-coronavirus adaptation to human
ACE2. EMBO J. 2005, 24, 1634–1643.
45. Wu, K.; Peng, G.; Wilken, M.; Geraghty, R.J.; Li, F. Mechanisms of host receptor adaptation by
SARS coronavirus. J. Biol. Chem. 2012, 287, 8904–8911.
46. Ren, W.; Qu, X.; Li, W.; Han, Z.; Yu, M.; Zhou, P.; Zhang, S.Y.; Wang, L.F.; Deng, H.; Shi, Z.
Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and
SARS-like coronavirus of bat origin. J. Virol. 2008, 82, 1899–1907.
47. Jeffers, S.A.; Tusell, S.M.; Gillim-Ross, L.; Hemmila, E.M.; Achenbach, J.E.; Babcock, G.J.;
Thomas, W.D.; Thackray, L.B.; Young, M.D.; Mason, R.J.; et al. CD209L (L-SIGN) is a receptor
for severe acute respiratory syndrome coronavirus. Proc. Natl. Acad. Sci. U. S. A. 2004, 101,
15748–15753.
48. Jeffers, S.A.; Hemmila, E.M.; Holmes, K.V. Human coronavirus 229E can use CD209L
(L-SIGN) to enter cells. Adv. Exp. Med. Biol. 2006, 581, 265–269.
49. Yang, Z.-Y.; Huang, Y.; Ganesh, L.; Leung, K.; Kong, W.-P.; Schwartz, O.; Subbarao, K.; Nabel,
G.J. pH-Dependent Entry of Severe Acute Respiratory Syndrome Coronavirus Is Mediated by the
Spike Glycoprotein and Enhanced by Dendritic Cell Transfer through DC-SIGN. J. Virol. 2004,
78, 5642–5650.
50. Han, D.P.; Lohani, M.; Cho, M.W. Specific asparagine-linked glycosylation sites are critical for
DC-SIGN- and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry. J. Virol.
2007, 81, 12029–12039.
51. Regan, A.D.; Whittaker, G.R. Utilization of DC-SIGN for entry of feline coronaviruses into host
cells. J. Virol. 2008, 82, 11992–11996.
52. Regan, A.D.; Ousterout, D.G.; Whittaker, G.R. Feline lectin activity is critical for the cellular
entry of feline infectious peritonitis virus. J. Virol. 2010, 84, 7917–7921.
Viruses 2012, 4
1030
53. Zhang, Y.; Buckles, E.; Whittaker, G.R. Expression of the C-type lectins DC-SIGN or L-SIGN
alters host cell susceptibility for the avian coronavirus, infectious bronchitis virus. Vet. Microbiol.
2012, 157, 285–293.
54. Nash, T.C.; Buchmeier, M.J. Entry of mouse hepatitis virus into cells by endosomal and
nonendosomal pathways. Virology 1997, 233, 1–8.
55. Matsuyama, S.; Taguchi, F. Receptor-induced conformational changes of murine coronavirus
spike protein. J. Virol. 2002, 76, 11819–11826.
56. Sturman, L.S.; Ricard, C.S.; Holmes, K.V. Conformational change of the coronavirus peplomer
glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell
fusion. J. Virol. 1990, 64, 3042–3050.
57. Zelus, B.D.; Schickli, J.H.; Blau, D.M.; Weiss, S.R.; Holmes, K.V. Conformational changes in the
spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine
CEACAM1 receptors or by pH 8. J. Virol. 2003, 77, 830–840.
58. Taguchi, F.; Matsuyama, S. Soluble receptor potentiates receptor-independent infection by murine
coronavirus. J. Virol. 2002, 76, 950–958.
59. Gallagher, T.M.; Escarmis, C.; Buchmeier, M.J. Alteration of the pH dependence of coronavirus-
induced cell fusion: Effect of mutations in the spike glycoprotein. J. Virol. 1991, 65, 1916–1928.
60. Qiu, Z.; Hingley, S.T.; Simmons, G.; Yu, C.; Das Sarma, J.; Bates, P.; Weiss, S.R. Endosomal
proteolysis by cathepsins is necessary for murine coronavirus mouse hepatitis virus type 2 spike-
mediated entry. J. Virol. 2006, 80, 5768–5776.
61. Eifart, P.; Ludwig, K.; Bottcher, C.; de Haan, C.A.; Rottier, P.J.; Korte, T.; Herrmann, A. Role of
endocytosis and low pH in murine hepatitis virus strain A59 cell entry. J. Virol. 2007, 81,
10758–10768.
62. Pu, Y.; Zhang, X. Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic
pathway independent of Eps15. J. Virol. 2008, 82, 8112–8123.
63. Chu, V.C.; McElroy, L.J.; Chu, V.; Bauman, B.E.; Whittaker, G.R. The avian coronavirus
infectious bronchitis virus undergoes direct low-pH-dependent fusion activation during entry into
host cells. J. Virol. 2006, 80, 3180–3188.
64. Yamada, Y.; Liu, D.X. Proteolytic activation of the spike protein at a novel RRRR/S motif is
implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus
infectious bronchitis virus in cultured cells. J. Virol. 2009, 83, 8744–8758.
65. Steinhauer, D.A. Role of hemagglutinin cleavage for the pathogenicity of influenza virus.
Virology 1999, 258, 1–20.
66. Leparc-Goffart, I.; Hingley, S.T.; Chua, M.M.; Jiang, X.; Lavi, E.; Weiss, S.R. Altered
pathogenesis of a mutant of the murine coronavirus MHV-A59 is associated with a Q159L amino
acid substitution in the spike protein. Virology 1997, 239, 1–10.
67. Gombold, J.L.; Hingley, S.T.; Weiss, S.R. Fusion-defective mutants of mouse hepatitis virus A59
contain a mutation in the spike protein cleavage signal. J. Virol. 1993, 67, 4504–4512.
68. de Haan, C.A.; Stadler, K.; Godeke, G.J.; Bosch, B.J.; Rottier, P.J. Cleavage inhibition of the
murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion.
J. Virol. 2004, 78, 6048–6054.
Viruses 2012, 4
1031
69. Hingley, S.T.; Leparc-Goffart, I.; Weiss, S.R. The spike protein of murine coronavirus mouse
hepatitis virus strain A59 is not cleaved in primary glial cells and primary hepatocytes. J. Virol.
1998, 72, 1606–1609.
70. Seidah, N.G.; Prat, A. The biology and therapeutic targeting of the proprotein convertases.
Nat. Rev. Drug Discov. 2012, 11, 367–383.
71. Seidah, N.G. The proprotein convertases, 20 years later. Methods Mol. Biol. 2011, 768, 23–57.
72. Simmons, G.; Gosalia, D.N.; Rennekamp, A.J.; Reeves, J.D.; Diamond, S.L.; Bates, P. Inhibitors
of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc. Natl. Acad. Sci.
U. S. A. 2005, 102, 11876–11881.
73. Simmons, G.; Reeves, J.D.; Rennekamp, A.J.; Amberg, S.M.; Piefer, A.J.; Bates, P.
Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike
glycoprotein-mediated viral entry. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 4240–4245.
74. Matsuyama, S.; Ujike, M.; Morikawa, S.; Tashiro, M.; Taguchi, F. Protease-mediated
enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci.
U. S. A. 2005, 102, 12543–12547.
75. Belouzard, S.; Chu, V.C.; Whittaker, G.R. Activation of the SARS coronavirus spike protein via
sequential proteolytic cleavage at two distinct sites. Proc. Natl. Acad. Sci. U. S. A. 2009, 106,
5871–5876.
76. Belouzard, S.; Madu, I.; Whittaker, G.R. Elastase-mediated activation of the SARS coronavirus
spike protein at discrete sites within the S2 domain. J. Biol. Chem. 2010, 285, 22758–22763.
77. Watanabe, R.; Matsuyama, S.; Shirato, K.; Maejima, M.; Fukushi, S.; Morikawa, S.; Taguchi, F.
Entry from the cell surface of severe acute respiratory syndrome coronavirus with cleaved S
protein as revealed by pseudotype virus bearing cleaved S protein. J. Virol. 2008, 82,
11985–11991.
78. Callan, R.J.; Hartmann, F.A.; West, S.E.; Hinshaw, V.S. Cleavage of influenza A virus H1
hemagglutinin by swine respiratory bacterial proteases. J. Virol. 1997, 71, 7579–7585.
79. Bosch, B.J.; Bartelink, W.; Rottier, P.J. Cathepsin L functionally cleaves the severe acute
respiratory syndrome coronavirus class I fusion protein upstream of rather than adjacent to the
fusion peptide. J. Virol. 2008, 82, 8887–8890.
80. Bottcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H.D.; Garten, W.; Matrosovich, M. Proteolytic
activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway
epithelium. J. Virol. 2006, 80, 9896–9898.
81. Shulla, A.; Heald-Sargent, T.; Subramanya, G.; Zhao, J.; Perlman, S.; Gallagher, T. A
transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus
receptor and activates virus entry. J. Virol. 2011, 85, 873–882.
82. Kam, Y.W.; Okumura, Y.; Kido, H.; Ng, L.F.; Bruzzone, R.; Altmeyer, R. Cleavage of the SARS
coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial
epithelial cells in vitro. PLoS One 2009, 4, e7870.
83. Glowacka, I.; Bertram, S.; Muller, M.A.; Allen, P.; Soilleux, E.; Pfefferle, S.; Steffen, I.; Tsegaye,
T.S.; He, Y.; Gnirss, K.; et al. Evidence that TMPRSS2 activates the severe acute respiratory
syndrome coronavirus spike protein for membrane fusion and reduces viral control by the
humoral immune response. J. Virol. 2011, 85, 4122–4134.
Viruses 2012, 4
1032
84. Bertram, S.; Glowacka, I.; Muller, M.A.; Lavender, H.; Gnirss, K.; Nehlmeier, I.; Niemeyer, D.;
He, Y.; Simmons, G.; Drosten, C.; et al. Cleavage and activation of the severe acute respiratory
syndrome coronavirus spike protein by human airway trypsin-like protease. J. Virol. 2011, 85,
13363–13372.
85. Belouzard, S.; Madu, I.; Whittaker, G.R. Elastase-mediated activation of the severe acute
respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain. J. Biol.
Chem. 2010, 285, 22758–22763.
86. Regan, A.D.; Shraybman, R.; Cohen, R.D.; Whittaker, G.R. Differential role for low pH and
cathepsin-mediated cleavage of the viral spike protein during entry of serotype II feline
coronaviruses. Vet. Microbiol. 2008, 132, 235–248.
87. Earp, L.J.; Delos, S.E.; Park, H.E.; White, J.M. The many mechanisms of viral membrane fusion
proteins. Curr. Top. Microbiol. Immunol. 2005, 285, 25–66.
88. Tamm, L.K.; Han, X. Viral fusion peptides: A tool set to disrupt and connect biological
membranes. Biosci. Rep. 2000, 20, 501–518.
89. Lai, A.L.; Li, Y.; Tamm, L.K. Interplay of proteins and lipids in virus entry by membrane fusion.
In Protein–Lipid Interactions; Tamm, L.K., Ed.; Wiley-VCH Verlag GmbH & Co. KGaA:
Weinheim, Germany, 2005; pp. 279–303.
90. Martin, I.; Ruysschaert, J.M. Common properties of fusion peptides from diverse systems.
Biosci. Rep. 2000, 20, 483–500.
91. Han, X.; Bushweller, J.H.; Cafiso, D.S.; Tamm, L.K. Membrane structure and fusion-triggering
conformational change of the fusion domain from influenza hemagglutinin. Nat. Struct. Biol.
2001, 8, 715–720.
92. Gomara, M.J.; Mora, P.; Mingarro, I.; Nieva, J.L. Roles of a conserved proline in the internal
fusion peptide of Ebola glycoprotein. FEBS Lett. 2004, 569, 261–266.
93. Durell, S.R.; Martin, I.; Ruysschaert, J.M.; Shai, Y.; Blumenthal, R. What studies of fusion
peptides tell us about viral envelope glycoprotein-mediated membrane fusion (review).
Mol. Membr. Biol. 1997, 14, 97–112.
94. Bosch, B.J.; Rottier, P.J. Nidovirus entry into cells. In Nidoviruses; Perlman, S., Gallagher, T.,
Snijder, E.J., Eds.; ASM Press: Washington, DC, USA, 2008; pp. 157–178.
95. Guillen, J.; Kinnunen, P.K.; Villalain, J. Membrane insertion of the three main membranotropic
sequences from SARS-CoV S2 glycoprotein. Biochim. Biophys. Acta. 2008, 1778, 2765–2774.
96. Guillen, J.; Perez-Berna, A.J.; Moreno, M.R.; Villalain, J. Identification of the membrane-active
regions of the severe acute respiratory syndrome coronavirus spike membrane glycoprotein using
a 16/18-mer peptide scan: Implications for the viral fusion mechanism. J. Virol. 2005, 79,
1743–1752.
97. Chambers, P.; Pringle, C.R.; Easton, A.J. Heptad repeat sequences are located adjacent to
hydrophobic regions in several types of virus fusion glycoproteins. J. Gen. Virol. 1990, 71,
3075–3080.
98. Petit, C.M.; Melancon, J.M.; Chouljenko, V.N.; Colgrove, R.; Farzan, M.; Knipe, D.M.;
Kousoulas, K.G. Genetic analysis of the SARS-coronavirus spike glycoprotein functional
domains involved in cell-surface expression and cell-to-cell fusion. Virology 2005, 341, 215–230.
Viruses 2012, 4
1033
99. Sainz, B., Jr.; Rausch, J.M.; Gallaher, W.R.; Garry, R.F.; Wimley, W.C. Identification and
characterization of the putative fusion peptide of the severe acute respiratory syndrome-associated
coronavirus spike protein. J. Virol. 2005, 79, 7195–7206.
100. Guillen, J.; Perez-Berna, A.J.; Moreno, M.R.; Villalain, J. A second SARS-CoV S2 glycoprotein
internal membrane-active peptide. Biophysical characterization and membrane interaction.
Biochemistry 2008, 47, 8214–8224.
101. Guillen, J.; de Almeida, R.F.; Prieto, M.; Villalain, J. Structural and dynamic characterization of
the interaction of the putative fusion peptide of the S2 SARS-CoV virus protein with lipid
membranes. J. Phys. Chem. B 2008, 112, 6997–7007.
102. Sainz, B., Jr.; Rausch, J.M.; Gallaher, W.R.; Garry, R.F.; Wimley, W.C. The aromatic domain of
the coronavirus class I viral fusion protein induces membrane permeabilization: Putative role
during viral entry. Biochemistry 2005, 44, 947–958.
103. Howard, M.W.; Travanty, E.A.; Jeffers, S.A.; Smith, M.K.; Wennier, S.T.; Thackray, L.B.;
Holmes, K.V. Aromatic amino acids in the juxtamembrane domain of severe acute respiratory
syndrome coronavirus spike glycoprotein are important for receptor-dependent virus entry and
cell-cell fusion. J. Virol. 2008, 82, 2883–2894.
104. Lu, Y.; Neo, T.L.; Liu, D.X.; Tam, J.P. Importance of SARS-CoV spike protein Trp-rich region in
viral infectivity. Biochem. Biophys. Res. Commun. 2008, 371, 356–360.
105. Guillen, J.; Moreno, M.R.; Perez-Berna, A.J.; Bernabeu, A.; Villalain, J. Interaction of a peptide
from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike
protein with phospholipid membranes. J. Phys. Chem. B 2007, 111, 13714–13725.
106. Madu, I.G.; Roth, S.L.; Belouzard, S.; Whittaker, G.R. Characterization of a highly conserved
domain within the severe acute respiratory syndrome coronavirus spike protein S2 domain with
characteristics of a viral fusion peptide. J. Virol. 2009, 83, 7411–7421.
107. Zhang, H.; Wang, G.; Li, J.; Nie, Y.; Shi, X.; Lian, G.; Wang, W.; Yin, X.; Zhao, Y.; Qu, X.;
et al. Identification of an antigenic determinant on the S2 domain of the severe acute respiratory
syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies. J. Virol.
2004, 78, 6938–6945.
108. Daniel, C.; Anderson, R.; Buchmeier, M.J.; Fleming, J.O.; Spaan, W.J.; Wege, H.; Talbot, P.J.
Identification of an immunodominant linear neutralization domain on the S2 portion of the murine
coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional
structure. J. Virol. 1993, 67, 1185–1194.
109. Bernini, A.; Spiga, O.; Ciutti, A.; Chiellini, S.; Bracci, L.; Yan, X.; Zheng, B.; Huang, J.; He,
M.L.; Song, H.D.; et al. Prediction of quaternary assembly of SARS coronavirus peplomer.
Biochem. Biophys. Res. Commun. 2004, 325, 1210–1214.
110. Bale, S.; Liu, T.; Li, S.; Wang, Y.; Abelson, D.; Fusco, M.; Woods, V.L., Jr.; Saphire, E.O. Ebola
virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.
PLoS Negl. Trop. Dis. 2011, 5, e1395.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).